HBsAg Secreting Hepatocyte-specific Expression Of Human Interferon Gamma Controls Hepatitis B Virus Replication In Vitro

Interferon gamma (IFNy) plays an important role in host defense against hepatitis virus infection. IFNy leads to a remarkable reduction of hepatitis B virus replication by inhibiting intrahepatic replication intermediates and eliminating pregenomic RNA from the hepatocyte through a noncytolytic mechanism. These effects of IFNγhave been demonstrated in hepatocytes and in HBV-transgenic or HBV-infected hosts such as mice, ducks and chimpanzees. However, if the therapy for HBV-infected patients is based on systemic administration of recombinant human IFNy, the higher blood IFNy levels are needed to maintain therapeutic IFNy levels in the liver, which often cause severe side effects. Therefore, gene therapy based on liver-specific and regulated delivery of IFNγmay provide a solution.HBV infection remains a major public health problem in China. The carrier rate of HBsAg is estimated to be as high as 10% of Chinese population which more than 1/3 of the carriers all over the world and a substantial proportion 75-95% of the primary liver cancer incidence is associated with this. The over expression of HBsAg is one of the major reason for degeneration and necrosis of HBV-infected hepatocytes since the gene integration of fragments HBV S, X and enhancer I in human genome is more than other gene fragments of HBV and especially for the strong ability of HBV S promoter to initiate transcription. According to this reason, we constructed the regulated IFNy expression vector targeting on the HBsAg secreting hepatocytes. With HBV S as the targeted gene and IFNγas the desired one, using self-regulating gene expression vector, we elevated the amount of liver-spcific expression of IFNγand controlled the replication of HBV through a noncytolytic mechanism. As a result of the regulated and HBsAg secreting hepatocyte-specific gene expression, the amount of IFNγexpression around the uninfected hepatocytes wiil not increase, and at the same time, IFNγexpression in HBsAg secreting hepatocytes will not be in an uncontrolled and long-lasting state. Therefore, the host defense system won't be activated so vigorously, leading to the death of a large number of hepatocytes and the occurrence of the fulminant hepatitis.Part One Construction of the HBsAg secreting hepatocyte-specific IFNy eukaryotic expression vector (pcDNA3.1-SCⅡ)Objective:Previously, we have reported the construction of a dicistronic expression vector containing the hepatitis C virus (HCV) internal ribosome entry site (IRES) as an effective promoter. To develop an efficient and non-viral gene delivery system based on the pcDNA3.1 vector that mediates the IFNy expression in hepatocyte-specific fashion for HBV gene therapy, we constructed the dicistronic eukaryotic expression vector containing the fragment of HCV IRES mentioned above and containing HBV surface gene as the targeted gene and the IFNγgene as the desired one.Methods:Using the approach described previously, we constructed the dicistronic expression vector with the biologically active fragment of HCV IRES based on the pcDNA3.1 vector. A fragment of HBV anti-surface gene, followed by HCV core protein gene, was cloned into the pcDNA3.1 vector before the HCV IRES site and the IFNγgene was also cloned into it following the HCV IRES site. In this way, we developed a dicistronic expression vector containing the fragments of HBV anti-surface gene and HCV core protein gene under the control of cytomegalovirus (CMV) promoter and expressing IFNγunder the control of HCV IRES downstream in turn. This vector system can be named HBV-anti S/HCV-core protein-IRES-IFNγor simply pcDNA3.1-SCⅡ. When HBsAg is secreted in the HBV-infected hpatocytes, the HBV anti-surface gene mRNA transcripts drove by the CMV promoter in the vector will be in dimerization with the mRNA transcripts of HBV S gene. Then the dimer before the initiation codon (AUG) of HCV core protein can inhibit the expression of HCV core protein and thus relieve the restriction of HCV core protein on the function of HCV IRES which finally enhances the expression of IFNγdrove by the HCV IRES. In this way, IFNγwill be expressed as a result of the existence of HBsAg secreted by the HBV-infected hepatocytes and whereas IFNy won't be expressed without the existence of HBsAg in primary hepatocytes.Results:The four gene fragments of the vector pcDNA3.1-SCⅡ, HBV anti S, HCV core protein, HCV IRES and IFNγobtained through PCR or digestion were ligated into T vector respectively for identification by PCR, restriction endonuclease analysis and DNA sequencing which showed that the sequences of the four gene fragments all had high homology (>96%) with corresponding sequence on GenBank. The results of the digestion and sequencing of the vector pcDNA3.1-SCⅡshowed that the four fragments were successfully inserted into the pcDNA3.1 vector.Conclusion:The recombinant vector pcDNA3.1-SCⅡwas successfully constructed through preliminary identification by restriction endonuclease analysis and DNA sequencing. Part Two Cell-specific expression of IFN-γin vitro following the vector pcDNA3.1-SCⅡObjective:To evaluate whether the HBsAg secreting hepatocyte-specific expression of IFN-γcan be efficiently mediated by pcDNA3.1-SCⅡin vitro.Methods:(1) We replaced IFNγgene in pcDNA3.1-SCⅡwith GFP gene, isolated by digestion from pEGFP-N1 plasmid, to construct the vector system HBV-anti S/HCV core-IRES-GFP, which can be simply named pcDNA3.1-SCⅠGFP. The pcDNA3. 1-SCⅠGFP was transfected into HepG2 and HepG2.2.15 cells with Lipofectamine 2000 respectively. The expressed fusion protein was examined by fluorescence microscopy after 48h of transfection. The recombinant plasmid pcDNA3.1-GFP was also constructed and was used to transfected into the two cell lines mentioned above. The transfection efficiency mediated by pcDNA3.1 was analyzed by monitoring the GFP expression through fluorescence microscopy. (2) The different doses of recombinant vector pcDNA3.1-SCⅡ(4μg,8μg and 16μg) was transfected into HepG2 and HepG2.2.15 cells according to the same method. The expression of IFN-γat protein level in the supernatant of the two cell lines was tested by ELISA after 24h, 48h,72h and 96h of transfection. The expression of IFN-γat mRNA level in the two cell lines was detected by reverse transcription PCR (RT-PCR) and was also detected by Western blot at protein level at 72h after transfection. (3) At 72h after transfection, HBV S mRNA expression in HepG2.2.15 cells transfected with 8μg pcDNA3.1-SCⅠGFP and 8μg pcDNA3.1-SCⅡwas measured by RT-PCR to evaluate the inhibitory ability of the vectors we constrcted on the expression of HBsAg at gene level. Cells non-trasnfected and only transfected with pcDNA3.1 vector were used as control. Results:(1) With the same transfection efficiency in the two cell lines, high expression of GFP mediated by pcDNA3.1-SCⅠGFP in HepG2.2.15 cells was monitored by fluorescence microscopy, whereas little expression of GFP in HepG2 cells was observed. (2) The Western blotting results indicated that expression of IFN-y was positive in HepG2.2.15 cells and negative in HepG2 cells after transduced with different doses of pcDNA3.1-SCⅡin the two cell lines at 72h after transfection. The corresponding results of the mRNA expression of IFN-γwas obtained through the detection of RT-PCR. In addition, the level of mRNA expression of IFN-γwas increased with the increase of the doses of pcDNA3.1-SCⅡ. The results obtained from the ELISA detection of the secreted IFN-γin the supernatant of the two cell lines showed further the expression of IFN-γin the supernatant of HepG2.2.15 cells was much more efficient than that in the supernatant of HepG2 cells at different transfection doses and times. A dose-dependent effect was also observed in the expression of IFN-y in the supernatant of HepG2.2.15 cells. In addition, maximum expression of IFN-y in the supernatant of HepG2.2.15 cells was obtained at 48h after transfection and no significant decrease was observed at 96h after transfection. The RT-PCR result of HBV S gene in HepG2.2.5 cells showed the expression of HBV S gene at mRNA level was significantly down-regulated following pcDNA3.1-SCⅠGFP and pcDNA3.1-SCⅡcompared with the non-trasnfected cells (P<0.05). There was no significant change of the level of HBV S mRNA in cells transfected with pcDNA3.1 vector compared with the non-trasnfected cells (P>0.05)Conclusion:The results indicated that pcDNA3.1-SCⅡhad the ability of mediating exogene expression specificlly in HBsAg secreting hepatocytes and inhibiting the expression of the targeted HBV S gene at mRNA level in vitro. Part Three Inhibitory effect of pcDNA3.1-SCⅡon replication and expression of hepatitis B virus in vitroObjective:To assess the inhibitory effect of pcDNA3.1-SCⅡon replication and expression of HBV at different doses of the vector and at different times after transfection and to evaluate the effect of pcDNA3.1-SCⅡat the highest dose on cell apoptosis using the HepG2.2.15 cells as the targeted cells.Methods:In order to assess the effect of pcDNA3.1-SCⅡon the replication and expression of HBV and the relationship between the effect of pcDNA3.1-SCⅡand its dosage, three different doses of pcDNA3.1-SCⅡ(4μg,8μg and 16μg) were transfected into HepG2.2.15 cells by using Lipofectin method transiently. HBsAg and HBeAg in the culture supernatant were detected by ELISA at 24h,48h,72h and 96h after transfection. Fluorescent quantitative PCR (FQ-PCR) was used to determine the level of HBV DNA in the culture supernatant of HepG2.2.15 cells at the four different times after transfection. In the experiments above, cells non-trasnfected were used as blank control, those transfected with control plasmid as negative control and those treated with 10μmol/l lamivudine as positive control. At 48h after transfection, Southern blot was performed to investigate the amount of replicative intermediates of HBV in cells. Effect of pcDNA3.1-SCⅡon apoptosis of HepG2.2.15 cells was analyzed by Annexin V-FITC/PI and flow cytometry at 96h after transfection. Cells treated with Lamivudine were used as positive control group and cells transfected with control plasmid were used as negative control group. Results:The level of the HBsAg, HBeAg and HBV DNA in the supernatant of HepG2.2.15 cells transfected with different doses of pcDNA3.1-SCⅡdecreased significantly compared with that of HepG2.2.15 cells without transfection. The inhibitory effect of pcDNA3.1-SCⅡon the level of HBsAg, HBeAg and HBV DNA in the cell supernatant increased with the increase of the doses of pcDNA3.1-SCⅡ(P<0.05) No significant difference was found between cells without transfection and cells transfected with control plasmid (P>0.05). The inhibitory effect of lamivudine on the HBV DNA in the cell supernatant was relatively strong, but the inhibitory effect was dramatically decreased on day 4. However, the inhibitory effect of pcDNA3.1-SCⅡon HBV DNA was still strong. The inhibitory effect of lamivudine on the secreting of HBsAg and HBeAg in supernatant were weaker than that of pcDNA3.1-SCⅡ. The inhibitory effect of lamivudine on HBeAg was intermediate between that of 4μg and 8μg pcDNA3.1-SCⅡand the inhibitory effect on HBsAg was weaker than that of 4μg pcDNA3.1-SCⅡ. The results of Southern blotting showed that the replicative intermediates of HBV in HepG2.2.15 cells could be efficiently inhibited following transfection of pcDNA3.1-SCⅡ, which was also in a dose-dependent manner. We observed that the most dramatic inhibitory effects of pcDNA3.1-SCⅡon viral expression (HBsAg and HBeAg) and replication (HBV DNA) were displayed at 48h after transfection and the inhibitory effect on HBsAg was stronger. In the cell apoptosis experiment, the apoptosis rate of HepG2.2.15 cells treated with Lamivudine was significantly higher than that of control at 96h after transfection (P<0.05) whereas no significant difference of the apoptosis rate was observed between the cells transfected with pcDNA3.1-SCⅡat the highest dose (16μg) and the control group (P>0.05)Conclusion:This gene delivery system (pcDNA3.1-SCⅡ) was capable of inhibiting HBV expression and replication in HepG2.2.15 cells efficiently without causing significant cell apoptosis.